Involvement of the Kinin B1 Receptor in Increased Permeability of Cerebral Microvessels in Rats Subjected to Autoimmune Encephalomyelitis.

Kinins are vasoactive peptides that are involved in various cellular mechanisms, including the inflammatory response. Kinins, released in vessel walls, exacerbate inflammation by modulating the production and release of pro-inflammatory factors via two types of G protein-related receptors-B1 and B2 receptors. B1 R is overexpressed during the inflammation that accompanies numerous neurological disorders, including multiple sclerosis (MS), in which loss of BBB integrity is an early pathomechanism of the disease. In this work, we apply pharmacological inhibition of the kinin B1 receptor with DALBK to investigate its effect on blood-brain barrier (BBB) permeability during the course of EAE, an animal model of MS. Functional, ultrastructural and molecular analyses were performed. The expression of selected BBB-associated proteins such as occludin and claudin-5 was assessed, as well as the astrocytic marker GFAP. We show that administration of a specific antagonist attenuates neurological symptoms in EAE rats and recovers the downregulation of TJ proteins and BBB leakage observed during the course of the disease, as well as significantly reducing the disease-specific activation of astroglia. The results show that B1 R-mediated signaling is involved in inducing molecular changes at the level of cerebral microvessels, leading to increased permeability of the BBB following neuroinflammation in EAE.

Kinin B1 receptor deficiency promotes enhanced adipose tissue thermogenic response to ß3-adrenergic stimulation.

OBJECTIVE AND DESIGN: Kinin B1 receptor (B1R) has a key role in adipocytes to protect against obesity and glycemic metabolism, thus becoming a potential target for regulation of energy metabolism and adipose tissue thermogenesis. MATERIAL OR SUBJECTS: Kinin B1 knockout mice (B1KO) were subjected to acute induction with CL 316,243 and chronic cold exposure. METHODS: Metabolic and histological analyses, gene and protein expression and RNA-seq were performed on interscapular brown adipose tissue (iBAT) and inguinal white adipose tissue (iWAT) of mice. RESULTS: B1KO mice, under acute effect of CL 316,243, exhibited increased energy expenditure and upregulated thermogenic genes in iWAT. They were also protected from chronic cold, showing enhanced non-shivering thermogenesis with increased iBAT mass (~ 90%) and recruitment of beige adipocytes in iWAT (~ 50%). Positive modulation of thermogenic and electron transport chain genes, reaching a 14.5-fold increase for Ucp1 in iWAT. RNA-seq revealed activation of the insulin signaling pathways for iBAT and oxidative phosphorylation, tricarboxylic acid cycle, and browning pathways for iWAT. CONCLUSION: B1R deficiency induced metabolic and gene expression alterations in adipose tissue, activating thermogenic pathways and increasing energy metabolism. B1R antagonists emerge as promising therapeutic targets for regulating obesity and associated metabolic disorders, such as inflammation and diabetes.

Kinin B1 receptor and TLR4 interaction in inflammatory response.

OBJECTIVE: We aimed to broaden our understanding of a potential interaction between B1R and TLR4, considering earlier studies suggesting that lipopolysaccharide (LPS) may trigger B1R stimulation. METHODS: We assessed the impact of DBK and LPS on the membrane potential of thoracic aortas from C57BL/6, B1R, or TLR4 knockout mice. Additionally, we examined the staining patterns of these receptors in the thoracic aortas of C57BL/6 and in endothelial cells (HBMEC). RESULTS: DBK does not affect the resting membrane potential of aortic rings in C57BL/6 mice, but it hyperpolarizes preparations in B1KO and TLR4KO mice. The hyperpolarization mechanism in B1KO mice involves B2R, and the TLR4KO response is independent of cytoplasmic calcium influx but relies on potassium channels. Conversely, LPS hyperpolarizes thoracic aorta rings in both C57BL/6 and B1KO mice, with the response unaffected by a B1R antagonist. Interestingly, the absence of B1R alters the LPS response to potassium channels. These activities are independent of nitric oxide synthase (NOS). While exposure to DBK and LPS does not alter B1R and TLR4 mRNA expression, treatment with these agonists increases B1R staining in endothelial cells of thoracic aortic rings and modifies the staining pattern of B1R and TLR4 in endothelial cells. Proximity ligation assay suggests a interaction between the receptors. CONCLUSION: Our findings provide additional support for a putative connection between B1R and TLR4 signaling. Given the involvement of these receptors and their agonists in inflammation, it suggests that drugs and therapies targeting their effects could be promising therapeutic avenues worth exploring.

Electroacupuncture alleviates diabetes-induced mechanical allodynia and downregulates bradykinin B1 receptor expression in spinal cord dorsal horn.

OBJECTIVE: Diabetic neuropathic pain (DNP) is one of the most prevalent symptoms of diabetes. The alteration of proteins in the spinal cord dorsal horn (SCDH) plays a significant role in the genesis and the development of DNP. Our previous study has shown electroacupuncture could effectively relieve DNP. However, the potential mechanism inducing DNP's genesis and development remains unclear and needs further research. METHODS: This study established DNP model rats by intraperitoneally injecting a single high-dose streptozotocin; 2 Hz electroacupuncture was used to stimulate Zusanli (ST36) and Kunlun (BL60) of DNP rats daily from day 15 to day 21 after streptozotocin injection. Behavioral assay, quantitative PCR, immunofluorescence staining, and western blotting were used to study the analgesic mechanism of electroacupuncture. RESULTS: The bradykinin B1 receptor (B1R) mRNA, nuclear factor-κB p65 (p65), substance P, and calcitonin gene-related peptide (CGRP) protein expression were significantly enhanced in SCDH of DNP rats. The paw withdrawal threshold was increased while body weight and fasting blood glucose did not change in DNP rats after the electroacupuncture treatment. The expression of B1R, p65, substance P, and CGRP in SCDH of DNP rats was also inhibited after the electroacupuncture treatment. CONCLUSION: This work suggests that the potential mechanisms inducing the allodynia of DNP rats were possibly related to the increased expression of B1R, p65, substance P, and CGRP in SCDH. Downregulating B1R, p65, substance P, and CGRP expression levels in SCDH may achieve the analgesic effect of 2 Hz electroacupuncture treatment.

Interaction of Angiotensin-(1-7) with kinins in the kidney circulation: Role of B1 receptors.

Changes in renal hemodynamics impact renal function during physiological and pathological conditions. In this context, renal vascular resistance (RVR) is regulated by components of the Renin-Angiotensin System (RAS) and the Kallikrein-Kinin System (KKS). However, the interaction between these vasoactive peptides on RVR is still poorly understood. Here, we studied the crosstalk between angiotensin-(1-7) and kinins on RVR. The right kidneys of Wistar rats were isolated and perfused in a closed-circuit system. The perfusion pressure and renal perfusate flow were continuously monitored. Ang-(1-7) (1.0-25.0 nM) caused a sustained, dose-dependent reduction of relative RVR (rRVR). This phenomenon was sensitive to 10 nM A-779, a specific Mas receptor (MasR) antagonist. Bradykinin (BK) promoted a sustained and transient reduction in rRVR at 1.25 nM and 125 nM, respectively. The transient effect was abolished by 4 µM des-Arg9-Leu8-bradykinin (DALBK), a specific kinin B1 receptor (B1R) antagonist. Accordingly, des-Arg9-bradykinin (DABK) 1 µM (a B1R agonist) increased rRVR. Interestingly, pre-perfusion of Ang-(1-7) changed the sustained reduction of rRVR triggered by 1.25 nM BK into a transient effect. On the other hand, pre-perfusion of Ang-(1-7) primed and potentiated the DABK response, this mechanism being sensitive to A-779 and DALBK. Binding studies performed with CHO cells stably transfected with MasR, B1R, and kinin B2 receptor (B2R) showed no direct interaction between Ang-(1-7) with B1R or B2R. In conclusion, our findings suggest that Ang-(1-7) differentially modulates kinin's effect on RVR in isolated rat kidneys. These results help to expand the current knowledge regarding the crosstalk between the RAS and KKS complex network in RVR.

Role of kinin receptors in skin pigmentation.

Previous studies have shown that all kinin system is constitutively expressed in the normal and inflamed skin, with a potential role in both physiological and pathological processes. However, the understanding regarding the involvement of the kinin system in skin pigmentation and pigmentation disorders remains incomplete. In this context, the present study was designed to determine the role of kinins in the Monobenzone (MBZ)-induced vitiligo-like model. Our findings showed that MBZ induces higher local skin depigmentation in kinin receptors knockout mice (KOB1R, KOB2R and KOB1B2R) than in wild type (WT). Remarkably, lower levels of melanin content and reduced ROS generation were detected in KOB1R and KOB2R mice treated with MBZ. In addition, both KOB1R and KOB2R show increased dermal cell infiltrate in vitiligo-like skin, when compared to WT-MBZ. Additionally, lack of B1R was associated with greater skin accumulation of IL-4, IL-6, and IL-17 by MBZ, while KOB1B2R presented lower levels of TNF and IL-1. Of note, the absence of both kinin B1 and B2 receptors demonstrates a protective effect by preventing the increase in polymorphonuclear and mononuclear cell infiltrations, as well as inflammatory cytokine levels induced by MBZ. In addition, in vitro assays confirm that B1R and B2R agonists increase intracellular melanin synthesis, while bradykinin significantly enhanced extracellular melanin levels and proliferation of B16F10 cells. Our findings highlight that the lack of kinin receptors caused more severe depigmentation in the skin, as well as genetic deletion of both B1/B2 receptors seems to be linked with changes in levels of constitutive melanin levels, suggesting the involvement of kinin system in crucial skin pigmentation pathways.

Effects of Bradykinin B2 Receptor Ablation from Tyrosine Hydroxylase Cells on Behavioral and Motor Aspects in Male and Female Mice.

The kallikrein-kinin system is a versatile regulatory network implicated in various biological processes encompassing inflammation, nociception, blood pressure control, and central nervous system functions. Its physiological impact is mediated through G-protein-coupled transmembrane receptors, specifically the B1 and B2 receptors. Dopamine, a key catecholamine neurotransmitter widely distributed in the CNS, plays a crucial role in diverse physiological functions including motricity, reward, anxiety, fear, feeding, sleep, and arousal. Notably, the potential physical interaction between bradykinin and dopaminergic receptors has been previously documented. In this study, we aimed to explore whether B2R modulation in catecholaminergic neurons influences the dopaminergic pathway, impacting behavioral, metabolic, and motor aspects in both male and female mice. B2R ablation in tyrosine hydroxylase cells reduced the body weight and lean mass without affecting body adiposity, substrate oxidation, locomotor activity, glucose tolerance, or insulin sensitivity in mice. Moreover, a B2R deficiency in TH cells did not alter anxiety levels, exercise performance, or motor coordination in female and male mice. The concentrations of monoamines and their metabolites in the substantia nigra and cortex region were not affected in knockout mice. In essence, B2R deletion in TH cells selectively influenced the body weight and composition, leaving the behavioral and motor aspects largely unaffected.

TRPV4 Activation and its Intracellular Modulation Mediated by Kinin Receptors Contribute to Painful Symptoms Induced by Anastrozole.

Anastrozole, an aromatase inhibitor, induces painful musculoskeletal symptoms, which affect patients' quality of life and lead to therapy discontinuation. Efforts have been made to understand the mechanisms involved in these painful symptoms to manage them better. In this context, we explored the role of the Transient Receptor Potential Vanilloid 4 (TRPV4), a potential transducer of several nociceptive mechanisms, in anastrozole-induced musculoskeletal pain in mice. Besides, we evaluated the possible sensibilization of TRPV4 by signalling pathways downstream, PLC, PKC and PKCε from kinin B2 (B2R) and B1 (B1R) receptors activation in anastrozole-induced pain. Anastrozole caused mechanical allodynia and muscle strength loss in mice. HC067047, TRPV4 antagonist, reduced the anastrozole-induced mechanical allodynia and muscle strength loss. In animals previously treated with anastrozole, the local administration of sub-nociceptive doses of the TRPV4 (4α-PDD or hypotonic solution), B2R (Bradykinin) or B1R (DABk) agonists enhanced the anastrozole-induced pain behaviours. The sensitizing effects induced by local injection of the TRPV4, B2R and B1R agonists in animals previously treated with anastrozole were reduced by pre-treatment with TRPV4 antagonist. Furthermore, inhibition of PLC, PKC or PKCε attenuated the mechanical allodynia and muscle strength loss induced by TRPV4, B2R and B1R agonists. The generation of painful conditions caused by anastrozole depends on direct TRPV4 activation or indirect, e.g., PLC, PKC and PKCε pathways downstream from B2R and B1R activation. Thus, the TRPV4 channels act as sensors of extracellular and intracellular changes, making them potential therapeutic targets for alleviating pain related to aromatase inhibitors use, such as anastrozole.

Evaluation of Novel B1R/B2R Agonists Containing TRIOZAN™ Nanoparticles for Targeted Brain Delivery of Antibodies in a Mouse Model of Alzheimer Disease.

The blood-brain barrier (BBB) is a major obstacle to the development of effective therapeutics for central nervous system (CNS) disorders, including Alzheimer's disease (AD). This has been particularly true in the case of monoclonal antibody (mAbs) therapeutic candidates, due to their large size. To tackle this issue, we developed new nanoformulations, comprising bio-based Triozan polymers along with kinin B1 and B2 receptor (B1R and B2R) peptide agonist analogues, as potent BBB-permeabilizers to enhance brain delivery of a new anti-C1q mAb for AD (ANX005). The prepared B1R/B2R-TRIOZAN™ nanoparticles (NPs) displayed aqueous solubility, B1R/B2R binding capacity and uniform sizes (~130-165 nm). The relative biodistribution profiles of the mAb loaded into these NPs versus the naked mAb were assessed in vivo through two routes of administrations (intravenous (IV), intranasal (IN)) in the Tg-SwDI mouse model of AD. At 24 h post-administration, brain levels of the encapsulated mAb were significantly increased (up to 12-fold (IV) and 5-fold (IN), respectively) compared with free mAb in AD brain affected regions, entorhinal cortex and hippocampus of aged mice. Liver uptakes remained relatively low with similar values for the nanoformulations and free mAb. Our findings demonstrate the potential of B1R/B2R-TRIOZAN™ NPs for the targeted delivery of new CNS drugs, which could maximize their therapeutic effectiveness.

Kinin receptors regulate skeletal muscle regeneration: differential effects for B1 and B2 receptors.

OBJECTIVE AND DESIGN: After traumatic skeletal muscle injury, muscle healing is often incomplete and produces extensive fibrosis. Bradykinin (BK) reduces fibrosis in renal and cardiac damage models through the B2 receptor. The B1 receptor expression is induced by damage, and blocking of the kallikrein-kinin system seems to affect the progression of muscular dystrophy. We hypothesized that both kinin B1 and B2 receptors could play a differential role after traumatic muscle injury, and the lack of the B1 receptor could produce more cellular and molecular substrates for myogenesis and fewer substrates for fibrosis, leading to better muscle healing. MATERIAL AND METHODS: To test this hypothesis, tibialis anterior muscles of kinin receptor knockout animals were subjected to traumatic injury. Myogenesis, angiogenesis, fibrosis, and muscle functioning were evaluated. RESULTS: Injured B1KO mice showed a faster healing progression of the injured area with a larger amount of central nucleated fiber post-injury when compared to control mice. In addition, they exhibited higher neovasculogenic capacity, maintaining optimal tissue perfusion for the post-injury phase; had higher amounts of myogenic markers with less inflammatory infiltrate and tissue destruction. This was followed by higher amounts of SMAD7 and lower amounts of p-SMAD2/3, which resulted in less fibrosis. In contrast, B2KO and B1B2KO mice showed more severe tissue destruction and excessive fibrosis. B1KO animals had better results in post-injury functional tests compared to control animals. CONCLUSIONS: We demonstrate that injured skeletal muscle tissues have a better repair capacity with less fibrosis in the presence of B2 receptor and absence of B1 receptor, including better performances in functional tests.

Kinin B1 and B2 receptors mediate cancer pain associated with both the tumor and oncology therapy using aromatase inhibitors.

Pain caused by the tumor or aromatase inhibitors (AIs) is a disabling symptom in breast cancer survivors. Their mechanisms are unclear, but pro-algesic and inflammatory mediators seem to be involved. Kinins are endogenous algogenic mediators associated with various painful conditions via B1 and B2 receptor activation, including chemotherapy-induced pain and breast cancer proliferation. We investigate the involvement of the kinin B1 and B2 receptors in metastatic breast tumor (4T1 breast cancer cells)-caused pain and in aromatase inhibitors (anastrozole or letrozole) therapy-associated pain. A protocol associating the tumor and antineoplastic therapy was also performed. Kinin receptors' role was investigated via pharmacological antagonism, receptors protein expression, and kinin levels. Mechanical and cold allodynia and muscle strength were evaluated. AIs and breast tumor increased kinin receptors expression, and tumor also increased kinin levels. AIs caused mechanical allodynia and reduced the muscle strength of mice. Kinin B1 (DALBk) and B2 (Icatibant) receptor antagonists attenuated these effects and reduced breast tumor-induced mechanical and cold allodynia. AIs or paclitaxel enhanced breast tumor-induced mechanical hypersensitivity, while DALBk and Icatibant prevented this increase. Antagonists did not interfere with paclitaxel's cytotoxic action in vitro. Thus, kinin B1 or B2 receptors can be a potential target for treating the pain caused by metastatic breast tumor and their antineoplastic therapy.

Kinins and their B1 and B2 receptors as potential therapeutic targets for pain relief.

Kinins are endogenous peptides that belong to the kallikrein-kinin system, which has been extensively studied for over a century. Their essential role in multiple physiological and pathological processes is demonstrated by activating two transmembrane G-protein-coupled receptors, the kinin B1 and B2 receptors. The attention is mainly given to the pathological role of kinins in pain transduction mechanisms. In the past years, a wide range of preclinical studies has amounted to the literature reinforcing the need for an updated review about the participation of kinins and their receptors in pain disorders. Here, we performed an extensive literature search since 2004, describing the historical progress and the current understanding of the kinin receptors' participation and its potential therapeutic in several acute and chronic painful conditions. These include inflammatory (mainly arthritis), neuropathic (caused by different aetiologies, such as cancer, multiple sclerosis, antineoplastic toxicity and diabetes) and nociplastic (mainly fibromyalgia) pain. Moreover, we highlighted the pharmacological actions and possible clinical applications of the kinin B1 and B2 receptor antagonists, kallikrein inhibitors or kallikrein-kinin system signalling pathways-target molecules in these different painful conditions. Notably, recent findings sought to elucidate mechanisms for guiding new and better drug design targeting kinin B1 and B2 receptors to treat a disease diversity. Since the kinin B2 receptor antagonist, Icatibant, is clinically used and well-tolerated by patients with hereditary angioedema gives us hope kinin receptors antagonists could be more robustly tested for a possible clinical application in the treatment of pathological pains, which present limited pharmacology management.

Spectroscopic characterization and in vitro studies of biological activity of bradykinin derivatives.

Eleven multiple analogs of bradykinin-a peptide that is a natural ligand of B1 and B2 receptors but does not bind or activate the B1 receptor unless Arg9 is removed from the sequence by the action of carboxypeptidase N-were synthesized. Their biological activity was examined on T-REx cell lines expressing B1 or B2 receptors using the intracellular IP1 assay. The mRNA expression of B1R and B2R in the lysate of tumor cell lines, e.g., U87-MG (human astrocytoma), SHP-77 (human small cell lung cancer), and H4 (human brain glioma), was determined. For five B1R antagonists, adsorption at the liquid/solid interface (Au nanoparticles (AuNPs) served as the solid surface) was discussed in terms of the vibrations of molecular fragments (structural factors) responsible for the biological properties of these analogs.

Kinin B1 receptor modulates mitochondrial activity responsivity in fasting and voluntary exercise.

The Kallikrein-Kinin System (KKS) plays an important role in energy metabolism. We have previously described the importance of the kinin B1 receptor (B1R) in metabolism regulation. Considering that the liver manages the different energy demands of different body tissues, we combined two stressful conditions - fasting and voluntary exercise - to address how B1R may affect liver metabolism, focusing on mitochondrial function. AIMS: To investigate how the kinin B1 receptor (B1R) modulates mitochondrial activity under stress conditions, focusing on the rate of energy expenditure and shift in metabolism. MAIN METHODS: Wild-type and B1R-knockout (B1KO) male mice remained in a calorimetric cage with a wheel for 7 days; 48 h before euthanasia, half of the animals from both groups were submitted to fasting conditions. Mitochondrial activity, ketone bodies, and gene expression involving mitochondrial activity were evaluated. KEY FINDINGS: B1R modulates the mitochondrial activity under fasting and voluntary exercise, reducing the VO2 expenditure and HEAT. B1KO animals who exercised and underwent fasting did not have increased glucose levels, suggesting a preference for lipids as an energy source. Moreover, these animals displayed RER around 0.8, which indicates a ß-oxidation increment. Interestingly, the lack of B1R did not induce mitochondrial activity and biogenesis, suggesting interference in metabolism responsivity, a condition modulated by sirtuins under PGC-1α control. SIGNIFICANCE: B1R modulates mitochondrial respiratory control ratios, which suggests metabolic suppression, influencing hepatic metabolism and, consequently, energy homeostasis.

An overview of kinin mediated events in cancer progression and therapeutic applications.

Kinins are bioactive peptides generated in the inflammatory milieu of the tissue microenvironment, which is involved in cancer progression and inflammatory response. Kinins signals through activation of two G-protein coupled receptors; inducible Bradykinin Receptor B1 (B1R) and constitutive receptor B2 (B2R). Activation of kinin receptors and its cross-talk with receptor tyrosine kinases activates multiple signaling pathways, including ERK/MAPK, PI3K, PKC, and p38 pathways regulating cancer hallmarks. Perturbations of the kinin-mediated events are implicated in various aspects of cancer invasion, matrix remodeling, and metastasis. In the tumor microenvironment, kinins initiate fibroblast activation, mesenchymal stem cell interactions, and recruitment of immune cells. Albeit the precise nature of kinin function in the metastasis and tumor microenvironment are not completely clear yet, several kinin receptor antagonists show anti-metastatic potential. Here, we showcase an overview of the complex biology of kinins and their role in cancer pathogenesis and therapeutic aspects.

Role of Endothelial Kinin B1 Receptor on the Membrane Potential of Transgenic Rat Aorta.

The kinin receptors are classically involved in inflammation, pain and sepsis. The effects of the kinin B1 receptor agonist des-Arg9-bradykinin (DBK) and lipopolysaccharide (LPS) were investigated by comparing the membrane potential responses of aortic rings from transgenic rats overexpressing the kinin B1 receptor (B1R) in the endothelium (TGR(Tie2B1)) and Sprague Dawley (SD) rats. No difference in the resting membrane potential in the aorta's smooth muscle from the transgenic and SD rats was observed. The aorta rings from SD rats hyperpolarized only to LPS but not to DBK, whereas the aorta rings from TGR(Tie2B1) responded by the administration of both drugs. DBK and LPS responses were inhibited by the B1 receptor antagonist R715 and by iberiotoxin in both cases. Thapsigargin induced a hyperpolarization in the smooth muscle of SD rats that was not reversed by R715, but was reversed by iberiotoxin and this hyperpolarization was further augmented by DBK administration. These results show that the model of overexpression of vascular B1 receptors in the TGR(Tie2B1) rats represent a good model to study the role of functional B1 receptors in the absence of any pathological stimulus. The data also show that KCa channels are the final mediators of the hyperpolarizing responses to DBK and LPS. In addition, we suggest an interaction between the B1R and TLR4, since the hyperpolarization induced by LPS could be abolished in the presence of R715.

Are noscapine and raloxifene ligands of the bradykinin B2 receptor? An assessment based on the human umbilical vein contractility assay.

The centrally acting antitussive opiate derivative, noscapine, has been claimed to be a non-competitive bradykinin B2 receptor antagonist. Raloxifene, a selective estrogen receptor modulator, was predicted to bind the bradykinin B2 receptor and to exert a partial agonist activity. These intriguing claims suggest that new molecular scaffolds ("chemotypes") may be identified for small molecule ligands of kinin receptors and that some off-target effects of noscapine or raloxifene may be mediated by bradykinin B2 receptors. An established contractile bioassay for ligands of the bradykinin B2 receptor, the isolated human umbilical vein, was exploited to characterize the inhibitory effect of noscapine and raloxifene on the B2 receptor-mediated contractile response to bradykinin. Observed effects were compared with those of the peptide antagonist icatibant, a potent, selective and competitive B2 receptor antagonist. Our results indicate that neither noscapine (2.5 µM) nor raloxifene (20 µM) behave as B2 receptor antagonists in concentrations that vastly exceeded an effective concentration of the control antagonist, icatibant; further, none of these drugs had direct contractile effects. It is suggested that the previously reported B2 receptor inhibitory effect of noscapine, a putative sigma-receptor agonist, might result from an indirect physiological antagonism, while raloxifene did not appear to have any significant affinity for the B2 receptors.

Soluble CD13 induces inflammatory arthritis by activating the bradykinin receptor B1.

CD13, an ectoenzyme on myeloid and stromal cells, also circulates as a shed, soluble protein (sCD13) with powerful chemoattractant, angiogenic, and arthritogenic properties, which require engagement of a G protein-coupled receptor (GPCR). Here we identify the GPCR that mediates sCD13 arthritogenic actions as the bradykinin receptor B1 (B1R). Immunofluorescence and immunoblotting verified high expression of B1R in rheumatoid arthritis (RA) synovial tissue and fibroblast-like synoviocytes (FLSs), and demonstrated binding of sCD13 to B1R. Chemotaxis, and phosphorylation of Erk1/2, induced by sCD13, were inhibited by B1R antagonists. In ex vivo RA synovial tissue organ cultures, a B1R antagonist reduced secretion of inflammatory cytokines. Several mouse arthritis models, including serum transfer, antigen-induced, and local innate immune stimulation arthritis models, were attenuated in Cd13-/- and B1R-/- mice and were alleviated by B1R antagonism. These results establish a CD13/B1R axis in the pathogenesis of inflammatory arthritis and identify B1R as a compelling therapeutic target in RA and potentially other inflammatory diseases.

Peptide fragments of bradykinin show unexpected biological activity not mediated by B1 or B2 receptors.

BACKGROUND AND PURPOSE: Bradykinin (BK-(1-9)) is an endogenous nonapeptide involved in multiple physiological and pathological processes. Peptide fragments of bradykinin are believed to be biologically inactive. We have now tested the two major peptide fragments of bradykinin in human and animals. EXPERIMENTAL APPROACH: BK peptides were quantified by MS in male rats. NO release was quantified from human, mouse and rat cells loaded with DAF-FM. Rat aortic rings were used to measure vascular reactivity. Changes in BP and HR were measured in conscious male rats. To evaluate pro-inflammatory effects both vascular permeability and nociception were measured in adult mice. KEY RESULTS: BK-(1-7) and BK-(1-5) are produced in vivo from BK-(1-9). Both peptides induced NO production in all cell types tested. However, unlike BK-(1-9), NO production elicited by BK-(1-7) or BK-(1-5) was not inhibited by B1 or B2 receptor antagonists. BK-(1-7) and BK-(1-5) induced concentration-dependent vasorelaxation of aortic rings, without involvement of B1 or B2 receptors. Intravenous or intra-arterial administration of BK-(1-7) or BK-(1-5) induced similar hypotensive response in vivo. Nociceptive responses of BK-(1-7) and BK-(1-5) were reduced compared to BK-(1-9), and no increase in vascular permeability was observed for BK-(1-9) fragments. CONCLUSIONS AND IMPLICATIONS: BK-(1-7) and BK-(1-5) are endogenous peptides present in plasma. BK-related peptide fragments show biological activity, not mediated by B1 or B2 receptors. These BK fragments could constitute new, active components of the kallikrein-kinin system.

In vitro pharmacological profile of PHA-022121, a small molecule bradykinin B2 receptor antagonist in clinical development.

PHA-022121 is a novel small molecule bradykinin B2 receptor antagonist, in clinical development for the treatment and prevention of hereditary angioedema attacks. The present study describes the in vitro pharmacological characteristics of PHA-022121 and its active metabolite, PHA-022484 (M2-D). In mammalian cell lines, PHA-022121 and PHA-022484 show high affinity for the recombinant human bradykinin B2 receptor with Ki values of 0.47 and 0.70 nM, respectively, and potent antagonism of the human bradykinin B2 receptor with Kb values of 0.15 and 0.26 nM, respectively (calcium mobilization assay). Antagonist potency at the recombinant cynomolgus monkey bradykinin B2 receptor is similarly high (Kb values of 1.42 and 1.12 nM for PHA-022121 and PHA-022484, respectively), however, potency at rat, mouse, rabbit and dog bradykinin B2 receptors is at least 100-fold lower than the potency at the human receptor for both compounds. In the human umbilical vein contractility assay, both PHA-022121 and PHA-022484 show a potent, surmountable and reversible B2 antagonist activity with pA2 values of 0.35 and 0.47 nM, respectively. The in vitro off-target profile of PHA-022121 and PHA-022484 demonstrates a high degree of selectivity over a wide range of molecular targets, including the bradykinin B1 receptor. It is concluded that PHA-022121 is a novel, low-molecular weight, competitive antagonist of the human bradykinin B2 receptor with high affinity, high antagonist potency, and high selectivity. It is about 20-fold more potent than icatibant at the human bradykinin B2 receptor as assessed using recombinant or endogenously expressed receptors.